PCR PRIMING FROM THE RESTRICTION-ENDONUCLEASE SITE 3' EXTENSION
PCR PRIMING FROM THE RESTRICTION-ENDONUCLEASE SITE 3' EXTENSION
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DOI：
10.1093/nar/21.20.4854
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发表时间：
1993-10-11
影响因子：
14.9
通讯作者：
HEALEY, A
中科院分区：
文献类型：
--
作者：
UPCROFT, P;HEALEY, A
在对肠原生动物寄生虫十二指肠贾第鞭毛虫（Giardia duodenalis）的耐药基因5'侧翼区域序列进行扩增的过程中，发现该区域在大肠杆菌中不稳定。作为克隆的替代方法，我们选择用Kalmanetal的连接-适配器方法进行PCR扩增。(1)，“vectorette”（剑桥研究生物化学）或“锚定pcr”系统之一（2,3,4）。连接-适配器原理取决于引物在远端限制性内切酶切割位点与模板的共价附着；因此，含有适当限制性内切酶5'延伸的双链连接引物被连接到模板上。使用已知的内部引物通过另一条链上的5'末端连接引物进行PCR扩增，从而产生一个互补副本，从中可以继续进行常规PCR扩增以产生未知区域的副本。对于内部引物，我们使用了我们已经测序并知道在大肠杆菌中稳定的基因克隆部分的双链DNA片段，类似于Sarkar和Sommer(5)描述的方法。然而，我们遇到了一些扩增伪影，其中包括产生明显非常高分子量的DNA和异常条带。这部分归因于双链DNA引物的自引（也见文献5），并且可以通过限制性内切酶切割从一端去除37 bp来防止。观察到异常的第二个原因是使用了从Kalman等人(1)所描述的改良的连接适配器，以合并SacI末端并连接到远端上游SacI限制位点。适配器具有3‘扩展，而不是Kalman等人描述的5’。这种组合的引物活性导致产生高分子量的DNA扩散，最终不会进入1%琼脂糖凝胶。边界条件的标准变化并不能阻止异常的发生。为了减少连接适配器明显过度的启动活性，我们省略了与模板的连接步骤，并最终省略了组合的非启动部分。添加只有4个碱基Sacd 3’同源性（AGCT）的单寡核苷酸引物（与内部引物一起）足以产生正确的PCR产物，而不存在先前观察到的伪像。这与内部引物(6)和重组(7)的基本3'端同源性要求是一致的。由于同源区域没有明显的异常结构或GC含量，该原理应广泛适用于其他限制性内切酶，这些酶只在PCR“行走”的远端引物位点产生少量的单链同源碱基对。复杂基因组PCR扩增的保真度最终取决于引物与模板的同源性（耦合到适当的边界条件下）和其他潜在目标位点的频率，特别是与引物的3'端（6,7）。我们在这里描述的方法原则上是由已知的内部序列的同源性所决定的，从这个内部序列到远端限制性位点特异性引物的扩增是被驱动的。潜在的伪产物，例如，从同一分子的两个末端SacI位点引物，可以通过与具有不同识别位点的第二个内切酶切割来减少，或者通过凝胶富集以获得预期大小的产物。到目前为止，我们还没有在基因组“行走”过程中遇到由单链sac特异性引物引起的伪像，并将其归因于单链的保持稳定性(8…
During the course of extending the sequence of the 5'flanking region of a gene involved indrug resistance from the intestinal protozoan parasite Giardia duodenalis, the region was found to be unstable in Escherichia coli. As an alternative to cloning we elected to amplify the region by PCR using the linker-adapter method of Kalmanetal.(1), one ofthe'vectorette'(Cambridge Research Biochemicals) or'anchored-PCR'systems (2, 3, 4). The linker-adapter principle depends upon the covalent attachment of the primer to the template at a distal restriction endonuclease cleavage site; the double-stranded linker-primer containing the appropriate restriction endonuclease 5'extension is therefore ligated to the template. A known internal primer is used to amplify by PCR back through the 5'terminus-ligated primer on the otherstrand, thereby generating a complementary copy from which conventional PCR amplification can be continued to generate copies of the unknown region. For the internal primer we utilised a double-stranded DNA segment from the cloned portion ofthe gene which we had already sequenced and knew was stable in E. coli, similar to the method described by Sarkar and Sommer (5). However, we encountered a number of amplification artifacts which included the generation of apparently very high molecular weight DNA and anomalous bands. This was partly ascribed to self-priming of the double stranded DNA primer (also see ref. 5) and could be prevented by removing 37 bp from one end by restriction endonuclease cleavage. A second cause of the observedanomalies was the use of thelinker-adapter, modified from that described by Kalman et al.(1) to incorporate a SacI terminus and ligated to a distal upstream SacIrestriction site. The adapter has a 3'extension instead of the 5'described by Kalman et al. The priming activityof this combination caused the generation of a high molecular weight spread of DNA which eventually would not enter a 1% agarose gel. Standard changesin boundary conditions did not preventthe anomalies. To reduce the apparent excessive priming activity of the linker-adapter we omitted the ligation step to the template, and eventually omitted the non-priming portion of the combination. The addition of the single oligonucleotide primer with only the 4 base Sacd 3'homology (AGCT) was sufficient (with the internal primer) to generate the correct PCR product free from the artifacts previously observed. This is consistent with essential 3'terminal homology requirements for internal priming (6) and recombination (7). Since the region of homology has no obviously unusual structure or GC content the principle should be widely applicable for other restriction endonucleases which create only a few base pairs of single-stranded homology for the distal priming site of PCR'walking'.The fidelity of PCR amplification from a complex genome ultimately depends upon the homology of the primers with the template (coupled to appropriate boundary conditions) and the frequency of other potential target sites, particularly with the 3'terminus of the primer (6, 7). The approach we havedescribed here is anchored in principle by the homology of the known internal sequence from which amplification to the distal restriction site-specific primer is driven. Potential artifacts, eg, priming from two terminal SacI sites in the same molecule could be reduced by cleavage with a second endonuclease having a different recognition site, or by gel enrichment for the expected size product. We have not encountered artifacts caused by the single-stranded Sac-specific primer during genomic'walking'thus far and attribute this to the single-stranded stacldng stability (8 …